C2 ENZYMATIC ACTIVITY
Enzymatic Activity of the Second Component of Complement? Neil R. Cooper*
ABSTRACT:
Isolated C2 and C2i preparations were able to hydrolyze a number of synthetic esters containing basic amino acids, among which N-a-acetylglycyl-L-lysine methyl ester (AcGlyLysOMe) was most susceptible. The cleaving activity was a property of the C2 molecule, since it correlated with the presence of C2 on analyses of C2 preparations by ultracentrifugation in sucrose gradients, filtration through Sephadex (3-200 columns, and on electrophoresis in acrylamide gels. Furthermore, acrylamide gel electrophoretic studies showed a shift in hydrolytic activity from the position occupied by C2 to that characteristic of C2i after incubation of C2 with C i s . The action was enzymati-
cally mediated as evidenced by a bell-shaped pH activity curve, a linear dependence on C2 concentration, and the presence of Michaelis-Menten kinetics. The Michaelis constant for cleavage of AcGlyLysOMe by C2 was 1.8 X l o w 2 mol. Cleavage of C2 by C i s increased C2 enzymatic activity, yet chemical oxidation of the molecule, although enhancing hemolytic activity, failed to increase C2 hydrolytic activity. The observed enzymatic activity of C2 was found to be relevant to the function of C2 in the C j ? complex, since AcGlyLysOMe competitively inhibited the Ca? mediated cleavage of C3 in free solution and the Ca2 dependent binding of C3 to cells.
T h e second component of complement ( ~ 2 is) an essential constituent of the key complement enzyme C42, or C3 convertase. Cd2 is generated from two inactive precursor molecules, C2 and C4, by the enzymatic action of C i s (MullerEberhard et al., 1967). The natural substrate of C42 is C3, which is cleaved by C42 into two fragments, C3a and C3b (Muller-Eberhard et al., 1967, Dias da Silva et al., 1967). The smaller fragment, C3a, has anaphylatoxin activity (Dias da Silva et al., 1967, Bokisch et al., 1969) while the larger fragment, C3b, interacts with C42 in an unknown manner to generate another proteolytic enzyme which has C5 as its substrate (Cochrane and Muller-Eberhard, 1968; S h i n et al., 1968). Several observations suggest that the enzymatic site of Ca2 resides in the C2 molecule. First, the ability to cleave C3 is acquired only on union of C2 with C4 and lost on dissociation of C2 from the complex (MullerEberhard et al., 1967). Second, the extent of Ca? activity is directly proportional to the amount of C2 employed to prepare the complex (Muller-Eberhard et al., 1966), and third, chemical oxidation of C2 prior to incorporation into Cd2 enhances the activity of the enzyme formed with the altered molecule (Muller-Eberhard et al., 1967). The present studies show that C2 is an enzyme able to hydrolyze certain synthetic esters containing lysine. The reaction exhibited Michaelis-Menten kinetics and a dependence on pH and C2 concentration. Furthermore, the ability of C4? to cleave C3 was competitively inhibited by a synthetic C2 substrate, indicating that the functional activity of Ca2 in the complement sequence is dependent on the enzymatic activity of C2.
Tris (Sigma Chemical Co., St. Louis, Mo.) were used to prepare buffers. Phenylmethanesulfonyl fluoride (PhCH2SOzF) and diisopropyl fluorophosphate (Dip-F) were purchased from Calbiochem, San Diego, Calif., and Boots Pure Drug Co., Ltd., Nottingham, England, respectively. Dithiothreitol and reagents for polyacrylamide gel electrophoresis were obtained from Bio-Rad Laboratories, Richmond, Calif. The following compounds were obtained from Cyclo Chemical, Los Angeles, Calif., or Schwarz/ Mann, Orangeburg, N.Y.:N-acetyl-L-arginine methyl ester (AcArgOMe), N-a-acetylglycyl-L-lysine methyl ester (AcGlyOMe), N-a-acetyl-L-lysine methyl ester (AcLysOMe), N-a-acetyl- L-ornithine methyl ester (AcOrnOMe), L-arginylglycine (Arg-Gly), L-arginyl-L-glutamic acid (Arg-Glu), L-arginyl-L-leucine (Arg-Leu), L-arginine methyl ester (ArgOMe), N-benzoyl-L-arginine methyl ester (BzArgOMe), N-benzoylglycine methyl ester (BzGlyOMe), N-a-benzoyl-L-lysine methyl ester (BzLysOMe), N-a-carbobenzoxy-L-argininemethyl ester (CbzArgOMe), N-a-carbobenzoxyglycine methyl ester (CbzGlyOMe), N-a-carbobenzoxy-L-lysine methyl ester (CbzLysOMe), L-lysyl-L-aspartic acid (Lys-Asp), L-lysine ethyl ester (LysOEt), L-lysyl-L-glutamic acid (Lys-Glu), L-lysyl-L-glutamyl-a-glycine (Lys-Glu-Gly), L-lysyl-L-leucinamide (Lys-Leu-amide), L-lysyl-L-a-lysine (Lys-Lys), L-lysine methyl ester (LysOMe), L-lysyl-L-serine (LysSer), N-a-tosyl-L-arginine methyl ester (Tos-ArgOMe), and N-a-tosyl-L-lysine methyl ester (Tos-LysOMe). The composition of Veronal-buffered saline containing 1.5 X M magnesium with or withM calcium and 5 X
'
Materials and Methods
Chemicals and Reagents. Reagent grade sodium phosphate (J.T. Baker Chemical Co., Phillipsburg, N.J.), sodium barbital (Sigma Chemical Co., St. Louis, Mo.), and ~
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From the Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037. Receiced February 24, 1975. This is publication number 934. This work was supported by U S . Public Health Service Program Grant AI-07007. * Recipient of U S . Public Health Service Research Career Development Award K04-AI-33630. +
Abbreviations used are: PhCH2SOZF, phenylmethanesulfonyl fluoride: Dip-F, diisopropyl fluorophosphate; AcArgOMe, N-acetyl-L-arginine methyl ester: AcGlyLysOMe, N-a-acetylglycyl-L-lysinemethyl ester; AcLysOMe, N-a-acetyl-L-lysine methyl ester; AcOrnOMe, N a-acetyl-L-ornithine methyl ester; ArgOMe, L-arginine methyl ester: BzArgOMe, N-benzoyl-L-arginine methyl ester; BzGlyOMe, N-benzoylglycine methyl ester: BzLysOMe, N-a-benzoyl-L-lysine methyl ester: CbzArgOMe, N-a-carbobenzoxy-L-arginine methyl ester; CbzGlyOMe, N-a-carbobenzoxyglycine methyl ester: CbzLysOMe, N-a-carbobenzoxy-L-lysine methyl ester; LysOEt, L-lysine ethyl ester: LysOMe. L-lysine methyl ester: Tos-ArgOMe, N-a-tosyl-l.-arginine methyl ester: Tos-LysOMe, N-a-tosyl-l.-lysine methyl ester. BIOCHEMISTRY,
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out 0.1% gelatin (GVB) has been described (Mayer, 1961). Carboxymethylated human albumin was prepared by a published method (Polley and Muller-Eberhard, 1968). Soybean and lima bean trypsin inhibitors were purchased from Worthington Biochemical Corp., Freehold, N.J. C1 inactivator, crI anti-trypsin, and anti-chymotrypsin were obtained through the courtesy of Drs. K . Drager and H . Schwick, Behringwerke AG, Marburg, Germany. Complement and Complement Reagents. Fresh human serum was employed for the isolation of C i s , C2, C3, and C4. C i s was isolated by a modification (Harpel and Cooper, 1975) of the previously published method (Valet and Cooper, 1974), while C 2 (Polley and Muller-Eberhard, 1968), C 3 (Nilsson and Muller-Eberhard, 1965), and C 4 (Schreiber and Muller-Eberhard, 1975) were isolated as has been described. C 2 was oxidized by treatment with iodine (Polley and Muller-Eberhard, 1967). Isolated C 3 was radiolabeled with lzSI by the method of McConahey and Dixon ( 1966). The cellular intermediates E A C 1, EAC 14, and EAC142 were prepared by published methods (Cooper and Muller-Eberhard, 1968; Cooper et al., 1970). Measurement of Complement Components. c i s , C2, C3, and C 4 were directly quantitated by using the Lowry technique (Lowry et al., 1951) with standard curves calibrated by micro Kjeldahl determinations. Specific hemolytic titrations were employed for the quantitative measurement of C2, C3, and C 4 activities (Cooper et al., 1970; Cooper and Muller-Eberhard, 1968, 1970). C i s activity was measured by incubating samples with 10 pg of isolated C 4 for 30 min a t 37' in a reaction volume of 50 p1 after which remaining hemolytically active C 4 was determined by effective molecule titration. Calibration curves relating Cis concentration to inactivation of C 4 were linear and 50% inactivation occurred with 1.6 ng of C i s . Formation of C2i. C2i was formed by incubation of C 2 with a 1 : l O O molar ratio of C i s for 30 min at 37'. C 2 hemolytic activity was measured to verify formation of C2i. Detection of C2 and C2i. C 2 and C2i in fractions from Sephadex (3-200 columns and sucrose density gradients and in eluates of acrylamide gels were detected by the Ouchterlony technique with monospecific antiserum to human C 2 (Polley and Muller-Eberhard, 1968). Generation of C22. C42 was formed after incubating 10 pg of C 4 and 1 1 pg of oxidized C 2 with 1 pg of Cis for 20 min at 37' in a total volume of 0.6 ml. After addition of Dip-F (see below) and additional incubation for 20 min a t 37', dilutions of the complex were incubated with 20 pg of C 3 in a reaction volume of 0.2 ml for 30 min a t 37' to determine the activity of the C;?j complex. Fifty percent inactivation of the hemolytic activity of C 3 was generally accomplished by 0. I ml of a 1 :40 dilution of the complex. For the study of the effect of AcGlyLysOMe on the C42-mediated cleavage of C3, the C42 preparation was diluted 20fold and 0.1-ml aliquots were incubated with varying amounts of C 3 in the presence or absence of AcGlyLysOMe as specified in the Results in a total reaction volume of 0.3 ml. Enzymatic Studies. All substances to be examined for enzymatic activity were incubated for 30 min a t 37' with 2 X M Dip-F added directly to the samples from a 5 M stock. The samples were then dialyzed overnight in the cold against a 0.1 M sodium phosphate buffer (pH 7.5) before incubation with substrates dissolved in the same buffer. Typical reaction mixtures consisted of 0.2 ml of enzyme solution and 50 pl of 5 X I O p 2 M substrate. Unless otherwise
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specified, the reactions mixtures were incubated for 2 hr a t 37' and then examined for cleavage by high voltage paper electrophoresis or the Siegelman technique for assay of methyl alcohol. High Voltage Paper Electrophoresis. Ten-microliter samples containing 0.1 p M of substrate were spotted on 46 X 57 cm sheets of Whatman Chromatography paper No. 1. The paper was moistened with pyridine acetate buffer (pH 6.4) and electrophoresis carried out to the cathode a t 1200 V for 75 min. The paper was dried a t 65" and stained with a solution of 2% ninhydrin in acetone. Quantitation of Methyl Alcohol. A slight modification of the Siegelman technique for measurement of methyl alcohol was employed (Siegelman et al., 1962); 50 pl of 0.75 M perchloric acid was added to a 250-pl sample containing 2.5 p h 4 of substrate. Next, 25 pl of 2% KMNOd followed by 25 pl of 10% Na2SO3 was added. The decolorized solutions were next incubated with 1 ml of 0.2% chromatopic acid (Eastman Kodak, Rochester, N.Y.) in 10.8 N HzS04 in a boiling water bath for 30 min. After the solution was cooled, 2 ml of HzO was added and the optical density of the solutions a t 580 nm determined. A standard methyl alcohol optical density curve was obtained from methanol standards included in each experiment. Polyacrylamide Gel Electrophoresis. Disc electrophoresis in 6% running gels was performed by the method of Davis (1964) in Tris-HCI-glycine buffer (pH 8.7). After electrophoresis, the gels were either stained with Amido Black or sectioned longitudinally and one-half was stained while the other half was sectioned at 2-mm intervals. The segments were placed in siliconized tubes and eluted with 50 pi of Tris-NaCI buffer (pH 7.5) containing 500 pg of carboxymethylated albumin/ml. Scanning of stained gels was performed on a Gilford linear transport device attached to a Gilford spectrophotometer (Gilford Instruments, Oberlin, Ohio). Sucrose Density Gradient Ultracentrifugation. Samples were sedimented in 7-3 1% linear sucrose density gradients prepared in 8 X 1 OW' M sodium phosphate, 1.5 X IO-' M NaCl and 1 X lo-' M EDTA (pH 5) in an SW 50.1 rotor a t 40,000 rpm for 18 hr in a Beckman L 2-50 ultracentrifuge. A constant ionic strength was maintained throughout the gradient. Chromatography on Sephadex G-200. Samples were filtered through 2 X 70 cm collumns of Sephadex C-200 in the pH 5.0 buffer described immediately above. Results
Cleucage of AcGlyLysOMe by C2. Highly purified preparations of human C 2 hydrolyzed several synthetic amino acid esters of which AcClyLysOMe was most susceptible. O n analysis of reaction mixtures by high voltage paper electrophoresis the ninhydrin reactive spot produced by AcGlyLysOMe partially or completely disappeared, and a new ninhydrin reactive product which had the same mobility as acetylglycyllysine appeared. Cleavage of the ester bond of AcGly LysOMe was confirmed by the Siegelman technique which showed that the action of C 2 on AcGlyLysOMe generated methyl alcohol. Representative results for cleavage of AcGlyLysOMe from separate experiments performed with seven different purified C 2 preparations a r e shown in Figure 1. In these experiments 3.6-1 1 pg of C 2 were incubated with 1 X IO-* M AcGlyLysOMe for 2 hr a t 37'. Although these studies were performed over a l-year period with preparations of C 2 which contained variable amounts
CZ E N Z Y M A T I C A C T I V I T Y
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F I G U R E I : Cleavage of N-a-acetylglycyl-L-lysine methyl ester (abhreviated AGLMe in figures)by C2 preparations. Seven different isolated C2 preparations were examined separately far their ability to hydrolyze AGLMe in 2 hr of incubation at 37'.
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F I G U R E 3: Acrylamide gel electrophoresis of C2 (upper three panels) and C2i (lower three panels). The upper three panels show the results obtained on testing the eluate^ of a gel to which 30 fig of DipF-treated C2 was applied far hemolytic activity, the stained aeryiamide gel, and an analysis of the ability of the eluates to cleave AGLMe as assessed by high voltage paper electrophoresis. A good correlation between the presence of CZ activity, the stained C2 protein hand. and ability to cleave AGLMc was obtained. The lower three panels show the results obtained with 30 Y Z of Dio-F-treated C2i. Considerable conversion of C2 to C2i which is devoid of hemolytic activity and a shift of AGLMe the psitian occupied by c 2 i are ~h~ heavy cleaving spot near the origin is due to glycine in the electrophoresis buffer. ~~~~~~~~~
F I G U R E 2: sucrose gradient ultracentrifugal analysis of C2 and a d y sis of fractions for C2 and ability lo cleave AGLMe: 50 P g of '22 M Dip-F and sedimented in a sucrose density treated with 2 X gradient. A good correlation was observed between the presence of C 2 hemolytic activity (upper panel), C2 protein by Ouchterlony analysis with anti-CZ (middle panel). and ?.hilily to cleave A G L M ~as determined by high voltage electrophoresis (lower panel).
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crose density gradient studies, the presence of C 2 protein and the ability to cleave AcClyLysOMe correlated well. Third. C 2 and C2i generated from C 2 bv treatment with C i s were subjected to electrophoresis in 6% polyacrylamide gels. Half of each gel was stained w h i k t h e remaining half was sectioned and eluted. The eluates were then examined for C2 hemolytic activity, for ability to cleave AcGlyLysOMe, and for the presence of active Cis. As depicted in Figure 3, the C 2 gel showed two bands, as is frequently observed with C 2 preparations (Polley and Muller-Eberhard. 1968). The heavier, cathodal band is characteristic of the mobility of C2, and C 2 hemolytic activity was eluted from segments corresponding to this band. The fainter, more anodal band had a mobility characteristic o f C 2 i which is generally present in varying proportions in C 2 preparations (Polley and Muller-Eberhard. 1968). Only eluates corresponding to the position of C 2 cleaved AcClyLysOMe (Figure 3). The other gel to which C2i had been applied showed a weaker band corresponding to the position of C 2 and a stronger band having the mobility characteristic of C2i. C 2 activity could not be eluted from this gel (Figure 3). AcClyLysOMe cleavage activity was now evident in eluates corresponding to the location of the C2i protein band. C i s activity was not detected in a n y of the eluates from either gel. Qualitatively identical results were obtained in three additional studies of this type with other C 2 preparations. In two of these studies, however, AcClyLysOMe hydrolyzing activity was present in two areas of the gel to which untreated C 2 had been applied, one of which correlated with the band produced by native C2, while the other corresponded to the C2i band. These studies thus indicate that AcClyLysOMe cleaving activity correlates with the presence of C 2 on sucrose density gradient ultracentrifugation, L
of CZi, a cleaved biologically inactive form of C 2 which has increased enzymatic activity as shown below, a fair relationship between protein concentration. and ability to hydrolyze AcGlyLysOMe was observed. Three types of studies were performed to show that hydrolytic activity was a property of the C 2 molecule and not due to a trypsin-like enzyme present as a contaminant in the C 2 preparations. First, C 2 was sedimented in sucrose density gradients in order to determine if there was a correlation between the location of C 2 and the ability to cleave AcGlyLysOMe. Since the C 2 isolation procedure does not include a separation based on size, it was thought that this technique might detect a potential contaminant. In the experiment depicted in Figure 2, which is representative of the four studies of this type, a good correlation was observed between the presence of C 2 protein, as assessed by Ouchterlony analysis, and the ability to cleave AcClyLysOMe, as determined by high voltage paper electrophoresis. Maximal hydrolytic activity, as shown by disappearance of the AcGlyLysOMe spot, occurred a t the midpoint of the fractions exhibiting reactivity with anti-C2. The heavier fractions reacting with a n t K 2 represented native C 2 as shown by the presence of hemolytic activity while the lighter fractions represented C2i, which has a lower sedimentation rate (Polley and Muller-Eberhard. 1968) and which is hemolytically inactive. Second, Dip-F-treated C 2 preparations were passed through Sephadex (3-200 columns and the fractions similarly assayed for C 2 hemolytic activity, C 2 protein, and ability to cleave AcClyLysOMe. As was observed in the su-
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COOPER
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Table I: Compounds Examined as Substrates for C 2 .
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F I G L R E 4: Effect of pH and ionic strength on the hydrolysis of AGLMe by C2.
Sephadex (3-200 filtration and acrylamide gel electrophoresis. Furthermore, in the acrylamide gel electrophoretic studies this activity shifted from the position occupied by C 2 to that characteristic of C2i after incubation with Cis. Characteristics of AcClyLysOMe Cleauage by C2. Severa1 parameters were studied in order to determine whether the cleavage of AcGlyLysOMe by C 2 was enzymatically mediated. As shown in Figure 4, the reaction was markedly dependent on pH, proceeding optimally a t p H 7.5, and somewhat dependent on ionic strength. A linear dose response curve to a t least 12 bg of C 2 was obtained for cleavage of AcGlyLysOMe. I n order to assess the effect of substrate concentration on hydrolysis, reaction mixtures containing a constant amount of C 2 and varying concentrations of AcGlyLysOMe were examined a t intervals for AcClyLysOMe cleavage. Zero-order kinetics were obtained for each substrate concentration examined and the velocity of the reaction decreased as the concentration of substrate increased. The values, when graphed in the manner of Lineweaver and Burk (1934), demonstrated that a linear relationship prevailed between the reciprocal of the initial velocity of AcGlyLysOMe cleavage and the reciprocal of the AcGlyLysOMe concentration. A Michaelis constant (K,) of 1.8 X lo-? M was calculated from the results. The above studies, taken together, indicate that C 2 enzymatically hydrolyzes AcGlyLysOMe. Substrate Specificity of the C2 Enzyme. In a series of experiments a number of potential substrates were incubated with C 2 or C2i isolated from purified preparations by sucrose density gradient ultracentrifugation. Analyses by high voltage paper electrophoresis revealed cleavage only of AcGlyLysOMe, CbzLysOMe, and AcyLysOMe (Table I ) . Quantitative measurements by the Siegelman technique for assay of methyl alcohol release were performed with a number of methyl esters containing arginine and lysine. As shown in Table I, AcGlyLysOMe was the most susceptible substrate followed by CbzLysOMe, Tos-LysOMe, CbzArgOMe, and AcLysOMe. Effect of Enzyme Inhibitors on C2 Enzymatic Activity The AcGlyLysOMe cleaving activity of 5 pg of isolated C 2 was not inhibited by previous incubation for 1 hr a t 37’ with 2 X 1 0-2 M Dip-F or PhCH>S02F, 100 fig of soybean or lima bean trypsin inhibitors, or by 200 g g of C1 inactivator, cy2 macroglobulin, cy1 anti-trypsin. or cy1 anti-chymotrypsin. Effect of C2 Cleavage on Enzymatic Activity. Cleavage of C 2 by C i s , a reaction termed activation, is necessary for formation of C a z and for progression of an ongoing complement reaction. The larger fragment of cleaved C 2 is able to bind to C 4 for a brief period of time after activation, thus generating Caz. Cleaved C 2 molecules which have lost the ability to bind to C 4 a r e termed C2i. Since the enzymatic activity of C 2 seemed increased after treatment with Cis,
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Pocitive AcGly LysOMe CbzLysOMe AcLysOMe Negative ArgOhle AcOrnOMe ArS-Gly .4rg- G1.U Arp-L eu LysOMe Ly90Et Ly\-Asp Lys-Glu Lys-Glu-Gly Lys-Leu-Amide Lys-Lys Lys-Ser ..
Siepelman Assay (pmol of MeOH)
AcGlyLysOMe CbzLysOMe Tos-LysOMe CbzArgOMr AcLysOhle AcArgOMe AcOrnOhfe BzArgOMe BzGlyOMe BzLysOMe CbzGylOMe LysOMe Tos-ArgOMe
0.65 0.43 0.18
0.10 0.09
